High capacity xylose transport in Candida intermedia PYCC 4715

Transcription

1 FEMS Yeast Research 3 (2003) 45^52 High capacity xylose transport in Candida intermedia PYCC 4715 Ma rk Ga rdonyi a;b,mafins Oº sterberg a;b;1, Carla Rodrigues a, Isabel Spencer-Martins a, Ba«rbel Hahn-Ha«gerdal b; a Centro de Recursos Microbiolo gicos (CREM), Faculty of Sciences and Technology, New University of Lisbon, Caparica, Portugal b Department of Applied Microbiology, Lund University, P.O. Box 124, Lund, Sweden Received 10 April 2002; received in revised form 3 June 2002; accepted 13 June 2002 First published online 3 August 2002 Abstract Xylose-utilising yeasts were screened to identify strains with high xylose transport capacity. Among the fastest-growing strains in xylose medium, Candida intermedia PYCC 4715 showed the highest xylose transport capacity. Maximal specific growth rate was the same in glucose and xylose media (W max = 0.5 h 31, 30 C). Xylose transport showed biphasic kinetics when cells were grown in either xylose- or glucose-limited culture. The high-affinity xylose/proton symport system (K m = 0.2 mm, V max = 7.5 mmol h 31 g 31 ) was more repressed by glucose than by xylose. The less specific low-affinity transport system (K = 50 mm, V max = 11 mmol h 31 g 31 ) appeared to operate through a facilitated-diffusion mechanism and was expressed constitutively. Inhibition experiments showed that glucose is a substrate of both xylose transport systems. ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords: Sugar transport; Hemicellulose; Xylose; Fermentation 1. Introduction * Corresponding author. Tel.: ; Fax: address: barbel.hahn-hagerdal@tmb.lth.se (B. Hahn-Ha«gerdal). 1 Present address: Microbiological Research Laboratory, Leo Pharmaceutical Products, 55 Industriparken, DK-2750 Ballerup, Denmark. An e cient conversion of the hemicellulose fraction in lignocellulosic material into ethanol requires the fermentation of xylose and other less abundant pentoses [1,2]. Wild strains of Saccharomyces cerevisiae, the fermentative yeast by excellence, lack the ability to ferment xylose. Natural xylose fermenters, like Pichia stipitis, depend on well-controlled oxygen-limited conditions for maximum ethanol production [3^5] and are highly susceptible to metabolic inhibitors present in lignocellulose hydrolysates [6], altogether preventing their industrial exploitation. Genes encoding the enzymes xylose reductase (XR) [7] and xylitol dehydrogenase (XDH) [8] from P. stipitis have been cloned and expressed in S. cerevisiae together with an overexpressed native xylulokinase [9,10]. The resulting recombinant strains are able to ferment xylose, even anaerobically, but at very low rates [10]. The poor performance has been attributed to glucose and xylose sharing common transport systems in S. cerevisiae, with a well-characterised facilitated-di usion mechanism and a nities for xylose about two orders of magnitude lower than for glucose [11,12]. Since glucose uptake is known to control, in large part, its fermentation rate by S. cerevisiae and related species [13,14], it is plausible that improving xylose transport in S. cerevisiae will lead to more e cient xylose-fermenting strains. The impact of the transport step on the metabolic ux may also be determined by the intracellular accumulation of the sugar given the relatively low a nity of XR for its substrate (K = 68 or 97 mm, depending on the cofactor [15]). Xylose/proton symporters occur in xylose-fermenting yeasts [16^21] and allow the intracellular accumulation of the pentose. These active transport systems show high a nities towards xylose and are rather speci c as compared to facilitated-di usion transporters. In a recent attempt to isolate the gene(s) responsible for active xylose uptake in P. stipitis [22], three genes coding for glucose transporters but with low a nity (K m = 49^145 mm) for xylose were found. The chances of isolating genes encoding higher-a nity xylose transporters would, in principle, increase when a yeast, which is able to express higher / 02 / $22.00 ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S (02)00137-X

2 46 M. Ga rdonyi et al. / FEMS Yeast Research 3 (2003) 45^52 levels of such a transporter under de ned growth conditions, were to be used as a source. To identify novel strains with potentially interesting xylose transporters, we screened a number of xylose-utilising yeasts. We provide here the results of the screening, describe the physiology of xylose metabolism in the selected Candida intermedia PYCC 4715 and present the kinetic characterisation of xylose and glucose transport in this strain as derived from both batch and chemostat cultures. 2. Materials and methods 2.1. Screening procedure Twenty-one xylose-assimilating yeast strains (Table 1), from the Portuguese Yeast Culture Collection (PYCC), were screened in this study. Their maximum speci c growth rates were determined, at 25 C with magnetic stirring, in 500-ml Erlenmeyer asks containing 200 ml Yeast Nitrogen Base (YNB) medium supplemented with 20 g l 31 xylose. The inoculum was prepared in the same medium from an overnight culture incubated in a rotary shaker. Those strains displaying more rapid growth were selected for xylose uptake rate measurements using cells harvested from the mid-exponential growth phase of either 20 g l 31 xylose or glucose medium. Uptake rates were estimated with 50 mm [ 14 C]xylose and by evaluating the presence of proton symport activity upon addition of 5 mm xylose to an aqueous cell suspension (see below) Culture conditions for C. intermedia PYCC 4715 Shake- ask cultures were performed in 1-l ba ed asks containing 200 ml de ned mineral medium [23] with either D-xylose or D-glucose (20 g l 31 ), at 30 C in a rotary shaker (150 rpm). Cells were harvested at mid-exponential phase (OD 620 = 0.6^0.9). Aerobic batch fermentation was conducted in computercontrolled glass bioreactors (Belach Bioteknik AB, Stockholm, Sweden) at 30 C and with ph controlled at 5.5 by automatic addition of NaOH (2 M). Aerobic conditions (po 2 s 40%) were maintained through air sparging and control of the stirring speed. Cultivation was performed in 800 ml de ned mineral media [23] supplemented with D- xylose (20 g l 31 ), D-glucose (20 g l 31 ) or a mixture of the two sugars (10 g l g l 31 ). The inoculum was prepared by transferring C. intermedia PYCC 4715, grown on slants with solid xylose medium, into 1-l Erlenmeyer asks containing 200 ml medium, ph 5.5, with either D- xylose (20 g l 31 ), for xylose cultures, or D-glucose (20 g l 31 ), for glucose cultures. In the case of the sugar mixture, D-glucose was used as substrate in the pre-culture. Cultures were incubated overnight at 30 C in a rotary shaker (150 rpm) and the cells harvested by centrifugation (6000 rpm, 5 min). Pellets were washed twice with NaCl (0.9%, w/v) and used to inoculate the batch culture to an initial OD 620 of 0.2. Sugar-limited chemostat cultures were conducted, at two dilution rates (0.11 h 31 and 0.40 h 31 ), in a Bio o III fermenter (New Brunswick Scienti c) with a working volume of 1.5 l, using de ned mineral medium [23] with either D-xylose (5 g l 31 )or D-glucose (5 g l 31 ) and antifoam (0.5 ml l 31 ) (Dow Corning Corporation). ph was controlled at 5.5 by automatic addition of NaOH (2 M) and dissolved oxygen was maintained above 40% by air sparging (1 l min 31 ) and a stirring speed of 350 rpm. The inoculum was prepared as for batch cultures except that the pre-culture was transferred directly, without centrifugation, to the fermenter Cell preparation for transport studies Cells were harvested by centrifugation (6000 rpm, 4 C), washed twice with ice-cold water and resuspended to a cell concentration of 30^50 g dry wt. l 31 in either ice-cold 100- mm Tris^citrate bu er (ph 5.0), for 14 C-sugar uptake, or ice-cold distilled water, to measure proton in ux. All cell suspensions were kept on ice C-Labelled sugar uptake Zero-trans in ux assays were performed, at 30 C, as previously described [24], using a reaction time of 5 s. D- [ 14 C]Xylose and -glucose were from Amersham International (Buckinghamshire, UK). Stock solutions of labelled xylose (about 500 cpm nmol 31 ) and glucose (about 1200 cpm nmol 31 ) at di erent concentrations were used to start the reactions. All experiments were conducted at least in duplicate. Kinetic constants of transport systems were estimated by non-linear regression analysis using commercial software (SigmaPlot) Sugar^proton symport activity Proton uptake rates were estimated by recording (input, 20 mv) the extracellular ph of an aqueous cell suspension before and after the addition of either xylose or glucose at di erent concentrations, using a ph meter (Radiometer, Copenhagen, Denmark) connected to a atbed recorder (Perkin-Elmer R100A). The cell suspension (200 Wl), freshly prepared, was mixed with 800 Wl distilled water in a temperature-controlled (30 C) reaction vessel and the initial ph was set to 4.95^5.05 using diluted HCl or NaOH. When it became apparent that no signi cant variation of the ph occurred with time, a baseline was recorded and 20 Wl of the sugar solution at the appropriate concentration were added. The system was calibrated using a known amount of HCl under the same reaction conditions.

3 M. Ga rdonyi et al. / FEMS Yeast Research 3 (2003) 45^ Analyses Cell growth was followed through the optical density of the culture at 620 nm. Substrate consumption and product formation were analysed and the cell dry weight determined as previously described [10]. Residual sugars were analysed on a HPAEC system with pulsed amperometric detection after separation on a Carbopac PA1 column (Dionex, Sunnyvale, CA, USA), using 16-mM NaOH as mobile phase (1 ml min 31 ) and a post-column addition of 300-mM NaOH (0.5 ml min 31 ). 3. Results 3.1. Screening Xylose is one of the carbon sources included in the battery of assimilation tests used for conventional yeast identi cation [25]. Based on semiquantitative physiological data available from the PYCC database, 21 xylose-assimilating (and glucose-fermenting) yeast strains, representing 10 di erent species, were screened for rapid growth in liquid mineral medium with xylose as the sole carbon and energy source. The maximum speci c growth rates, obtained at 25 C, varied between 0.14 h 31 and 0.37 h 31 (Table 1). In a second series of experiments, the xylose transport capacity in 12 of those strains, including the xylose-fermenting yeasts Candida shehatae [26], Pachysolen tannophilus [27] and P. stipitis [3,28], was estimated using a high concentration of labelled xylose. In parallel assays, nine of these strains were tested with respect to the presumptive presence of a xylose-proton symporter. A wide range of xylose uptake rates, 1.2^6.6 mmol h 31 (g 31 dw) 31, was obtained and in six strains a concomitant uptake of xylose and protons was observed. In all cases, no xyloseproton symport activity was detected in glucose-grown cells (not shown). As a result of this screening, C. intermedia PYCC 4715 emerged as the better performing strain, capable of rapid growth in xylose medium and showing the highest xylose transport capacity associated to the ability to transport xylose by an active transport system. Therefore, it was selected for further characterisation of xylose transport Growth characteristics of C. intermedia PYCC 4715 C. intermedia PYCC 4715 was grown under aerobic conditions in a high-performance bioreactor with xylose, glucose or a mixture of these sugars as sole carbon and energy source. Cell growth, sugar utilisation and product formation were followed along the incubation time. The maximum growth rates were found to be similar in glucose (0.50 h 31 ) and xylose (0.49 h 31 ) media. In the sugar mixture, cells started to consume xylose only when glucose was almost depleted ( 6 2gl 31 ). However, no di erence in growth rate and no lag phase were observed upon glucose depletion. Biomass was the only product when glucose was used as substrate. From xylose, a small amount (0.5 g l 31 ) of xylitol was also formed. Table 1 Speci c growth rate in xylose medium, initial uptake rates of labelled xylose (50 mm) and presence/absence of active xylose transport in selected yeast strains analysed in this study Species Strain (PYCC No.) W max (h 31 ) Initial uptake rate of D-xylose (mmol h 31 g 31 ) Candida boidinii n.d. n.d Candida friedrichii n.d. n.d Candida intermedia Candida membranifaciens n.d. n.d. Candida parapsilosis n.d. n.d. Candida shehatae n.d n.d. Debaryomyces hansenii n.d. n.d. Pachysolen tannophilus n.d. n.d. Pichia guilliermondii n.d. n.d n.d. n.d n.d. n.d n.d. Pichia stipitis n.d., not determined. Xylose/H þ symport activity

4 48 M. Ga rdonyi et al. / FEMS Yeast Research 3 (2003) 45^52 Fig. 1. Xylose uptake by C. intermedia PYCC 4715 grown in shake- asks. Eadie^Hofstee plots of initial uptake rates of (F) D-[ 14 C]xylose and of (b) initial proton in ux rates upon xylose addition to cells grown in 20 g l 31 xylose medium. R, initial uptake rates of D- [ 14 C]xylose by cells grown in 20 g l 31 glucose medium Kinetic characterisation of sugar uptake in C. intermedia PYCC 4715 Both xylose and glucose uptakes were kinetically characterised under di erent growth conditions. The kinetic constants were calculated using initial (5 s) uptake rates of labelled sugar in zero-trans-in ux experiments. The presence of sugar^proton symport activity was also investigated in unbu ered cell suspensions using a ph electrode. Table 2 summarises the kinetic constants determined for xylose and glucose uptake in shake- ask-grown cells as well as in chemostat-grown cells under sugar limitation. Fig. 2. Xylose uptake by C. intermedia PYCC 4715 grown in the chemostat (D = 0.11 h 31 ) under xylose limitation (F) and glucose limitation (R). Eadie^Hofstee plots of initial uptake rates of D-[ 14 C]xylose Xylose transport Cells of C. intermedia PYCC 4715 grown in shake- ask xylose cultures showed [ 14 C]xylose uptake following biphasic kinetics (Fig. 1). At low concentrations, xylose was mainly transported by a high-a nity system with low capacity. Low concentrations of glucose (0.25^0.50 mm) strongly inhibited this system in contrast to L-arabinose and D-ribose, which were weak inhibitors with inhibition constants over 20 mm (not shown). At higher concentrations, most of the xylose was taken up by a system with lower a nity but with much higher capacity. An apparent xylose/h þ symport, as estimated by initial proton in ux rates, followed kinetics similar to the high-a nity [ 14 C]xylose uptake (Fig. 1). In shake- ask glucose-grown cells, one transport system was found for xylose with kinetic constants comparable to those found for the low-a nity system of the xylosegrown cells (Fig. 1). No high-a nity component was observed and no proton in ux was detected upon xylose addition. Glucose (2.5 mm) inhibited xylose uptake under these conditions, but the type of inhibition was unclear due to the low xylose uptake rates. To investigate the regulation of the high-a nity xylose transport in more detail, C. intermedia PYCC 4715 was cultivated in a xylose-limited chemostat (Table 2). At low dilution rate (D = 0.11 h 31 ), the cells manifested a signi cantly increased capacity of the high-a nity xylose transport system although the a nity was similar to the value found in shake- ask-grown cells. A parallel increase in the proton in ux rates accompanying xylose uptake (not shown) supported these results. The presence of a lowa nity component became evident in the biphasic Ea- Table 2 Kinetic constants of xylose uptake in C. intermedia PYCC 4715 grown under di erent conditions Cultivation system Growth substrate Residual sugar concentration (mm) Xylose uptake K m (mm) V max (mmol h 31 g 31 ) Shake- ask Xylose K m1 = 0.28 þ 0.07 V max1 = 1.0 þ 0.1 K m2 = 51.5 þ 26.3 V max2 = 10.0 þ 3.6 Glucose K m = 51.9 þ 9.4 V max = 11.2 þ 1.4 Chemostat D = 0.11 h 31 Xylose 0.11 K m1 = 0.20 þ 0.04 V max1 = 7.5 þ 0.5 K m2 W50 V max2 (see text) Glucose K m1 = 0.22 þ 0.03 V max1 = 7.1 þ 0.2 K m2 W50 V max2 (see text) Chemostat D = 0.40 h 31 Xylose K m1 = 0.27 þ 0.07 V max1 = 3.2 þ 0.3 K m2 W50 V max2 (see text)

5 M. Ga rdonyi et al. / FEMS Yeast Research 3 (2003) 45^52 49 die^hofstee plots (Fig. 2). Non-linear regression analysis of the experimental data tted with a two-component Michaelis^Menten kinetics in which the low-a nity transport system would have a K of 50 mm and a V max of approximately 7 mmol h 31 (g dry wt.) 31. Glucose, at very low concentrations (0.04 mm), was a strong inhibitor of xylose uptake. When the dilution rate was increased to 0.4 h 31, the capacity of the high-a nity xylose uptake system decreased about 60%, the remaining characterisitics of xylose transport not di ering signi cantly. The kinetics of xylose transport in cells of C. intermedia PYCC 4715 harvested from a glucose-limited chemostat (D = 0.11 h 31 ) were similar to those observed in cells cultivated in the xylose-limited chemostat (Fig. 2). Glucose was a strong inhibitor, appearing to a ect both transport systems Glucose transport Glucose transport in shake- ask xylose-grown cells of C. intermedia PYCC 4715 followed biphasic kinetics (not shown). The high-a nity system, with K m = 0.1 mm and V max = 1.5 mmol h 31 (g dry wt.) 31, had a lower capacity than the low-a nity system with K m = 2.5 mm and V max = 5 mmol h 31 (g dry wt.) 31. The addition of 75 mm xylose almost abolished glucose uptake by the higha nity system and moderately inhibited transport by the low-a nity component. The activity of a putative glucose/ proton symporter was detected but could not be determined because of the rapid extracellular acidi cation following glucose addition to the aqueous cell suspension. In shake- ask glucose-grown cells only one transport system was observed for glucose (K m = 3.3 mm, V max = 7.7 mmol h 31 g 31 ; see Fig. 3). Glucose uptake was competitively inhibited by xylose with an inhibition constant of 54 þ 4 mm (Fig. 3). No proton in ux was detected upon addition of either xylose or glucose to the aqueous cell suspension. Fig. 3. Inhibition of glucose uptake by xylose. Eadie^Hofstee plots of initial uptake rates of D-[ 14 C]glucose by C. intermedia PYCC 4715 grown in 20 g l 31 glucose medium, without xylose (O) and in the presence of 75 mm xylose (P). Fig. 4. Initial uptake rates of D-[ 14 C]glucose by C. intermedia PYCC 4715 grown in the chemostat under xylose limitation at D = 0.11 h 31. In cells harvested from the xylose-limited chemostat operating at low dilution rate (D = 0.11 h 31 ), the uptake of labelled glucose did not follow Michaelis^Menten-type kinetics (Fig. 4). The initial rate of glucose transport increased with increasing glucose concentrations up to 0.1 mm, reaching an uptake rate of around 3.5 mmol h 31 (g dry wt.) 31. At higher concentrations the uptake rate decreased markedly until the concentration reached around 1 mm, above which the rate increased again with the glucose concentration. The behaviour was similar in cells grown under glucose limitation, except that the decrease in uptake rate started at a slightly higher glucose concentration. The same pattern was observed with cells grown in the xylose-limited chemostat at the higher dilution rate (0.4 h 31 ), although the decrease in uptake rate was not so pronounced. 4. Discussion The preliminary screening showed that the speci c growth rates of C. intermedia in xylose medium were consistently comparable to the values obtained with the xylose fermenter P. stipitis. Other yeasts tested, namely C. shehatae and strains of Pichia guilliermondii, displayed similar ability to grow in the same medium. However, the C. intermedia strains, and in particular C. intermedia PYCC 4715, exhibited signi cantly higher xylose transport activities rendering this glucose-fermenting yeast an interesting model to understand the role played by transport on xylose metabolism. Moreover, the presence of a xylose/h þ symporter provides an additional interesting characteristic since no gene encoding an active xylose transporter has been isolated from yeast so far. Growth kinetics and biomass yields in aerobic conditions were approximately the same in glucose and xylose medium, which con rmed the e ciency of C. intermedia PYCC 4715 as a xylose-assimilating yeast.

6 50 M. Ga rdonyi et al. / FEMS Yeast Research 3 (2003) 45^52 Xylose transport in yeasts has been thoroughly investigated at the biochemical level (reviewed in [29,30]), with a particular emphasis on P. stipitis for its potential applied importance as the best xylose fermenter. When studying the same strain, conclusions drawn by di erent groups on the number, nature and kinetic characteristics of operational transport mechanisms may appear as contradictory. The con icting data could, however, re ect di erent experimental conditions. In our experience, variables like the residual sugar concentration when harvesting the cells, the time between the preparation of the cell suspension and the e ective testing, the density of the cell suspension and, in particular, the reaction time have a strong in uence on the reproducibility of the results. Those studies, however, agree in showing that yeasts able to metabolise xylose generally produce a more or less speci c high-a nity xylose/proton symporter, functioning at relatively low sugar concentrations, and a facilitated-di usion transport system with one to two orders of magnitude higher a nity for glucose than for xylose. Xylose uptake in C. intermedia PYCC 4715 was studied in both shake- ask-grown cells and in cells grown in sugar-limited chemostat. The use of shake- ask-grown cells for uptake studies o ers a fast and easy way to obtain information about transport characteristics. However, in these studies the substrate concentration and other conditions change with time, not allowing an adequate elucidation of the mechanisms regulating the activities of sugar transport systems [31]. To ensure a constant supply of cells with a de ned physiological background we have also used sugar-limited chemostat cultures. Both xylose and glucose uptake by cells grown in shake- ask with xylose medium followed biphasic kinetics, indicating the existence of at least two distinct transport systems, similar to what has been found in other yeasts. During studies of polyol transport in a di erent strain of C. intermedia [32], glucose uptake was also found to follow biphasic kinetics, the glucose/h þ symporter being repressible by glucose and unspeci c since it accepted several polyols, hexoses and pentoses, including xylose, as substrates. The apparent K m of the high-a nity system in C. intermedia PYCC 4715 was around two orders of magnitude lower than the K m of the low-a nity system for xylose. In this respect, there is a striking resemblance to Pichia heedii [19]. Con rming the preliminary data obtained at the screening stage, the maximum xylose uptake rate was again signi cantly superior to the rates reported for the xylose fermenters C. shehatae [17] and P. stipitis [18], the di erence in shake- ask xylose-grown cells being due to the higher rates of the low-a nity transport system. Alkalisation upon either xylose or glucose addition to the unbu ered cell suspension indicated the presence of a sugar/proton symport activity. The kinetic constants determined for the xylose/proton symporter were similar to those of the high-a nity component as inferred from experiments with radiolabelled xylose, pointing to a xylose: proton stoichiometry of 1:1. It is worth mentioning that previous work in C. intermedia [32] indicated a glucose: proton stoichiometry of 1:2, whereas each sorbitol was cotransported, by the same transport sytem, with only one proton. This discrepancy may probably be ascribed to the use of tritiated glucose, instead of [ 14 C]glucose, in the labelled sugar uptake experiments reported by that author. The addition of glucose to the unbu ered cell suspension induced an initial alkalisation rapidly followed by a very fast acidi cation, believed to be due to a highly active membrane-bound H þ -ATPase, that ultimately prevented the determination of initial uptake rates based on proton in ux. The available data suggest that both xylose and glucose are transported by repressible high-a nity proton symporters (possibly a common active transport system, but that could not be clearly demonstrated experimentally as explained in the text) and low-a nity facilitated-di usion system(s). Growth in glucose medium completely repressed the proton symporter, as no alkalisation was detected upon addition of either glucose or xylose to cells harvested at the early exponential phase in shake- asks. Xylose acts as a weaker or partial repressor for the sugar/ proton symporter, indicated by the decrease in the V max associated with the increase in the residual xylose concentration in the fermenter (Table 2). Catabolite repression by xylose has been reported before in lamentous fungi [33]. In addition to catabolite repression, the high-a nity glucose transport is also subject to substrate inhibition, as observed in P. stipitis [18]. The rate of glucose uptake at substrate concentrations ranging from 0.1 to 1 mm decreased, increasing again for concentrations above 1 mm(fig. 4). This behaviour could possibly arise from the interplay between the active transport system, requiring metabolic energy to function, and the facilitated-di usion mechanism. Conceivably, the former would switch o as soon as the extracellular sugar concentration is high enough for the passive transport to operate e ciently. The cell would therefore modulate the activity of the existing transport systems according to its metabolic needs, thereby saving energy. The di erences in kinetic constants of the two transport systems would explain the response within the intermediate concentration range. Substrate inhibition of the glucose/h þ symporter, combined with the rapid acidi cation of aqueous cell suspensions upon glucose addition, did not allow us to obtain reliable gures for the kinetic constants of the high-a nity glucose transport system. Non-linear regression on the data points below 0.1 mm resulted in K m values between 0.09 and 0.17 mm, in agreement with the K m of 0.16 mm determined with another C. intermedia strain [32]. The capacity of the system was high (V max W7^9 mmol h 31 g 31 ) in cells obtained from the chemostat at low dilution rate and decreased by half in the high-dilution rate xylose-limited culture. The high-a nity xylose uptake was strongly inhibited by glucose that a ected both the K m and the V max of the

7 M. Ga rdonyi et al. / FEMS Yeast Research 3 (2003) 45^52 51 system. Non-competitive inhibition of the xylose/proton symport has been observed in C. shehatae [17] and P. stipitis [18] and it was explained by the sharing of protons as co-substrate of both transport systems. While this could justify the reduced V max, the increase in the K m might be due to glucose competing with xylose for the binding to the high-a nity xylose transporter. In contrast to glucose, pentose sugars (L-arabinose and D-ribose) had little e ect on the high-a nity xylose transport system. Xylose and glucose are mutual competitive inhibitors of their respective low-a nity uptake. Apparently, this is the only transport system present in C. intermedia PYCC 4715 grown in shake- asks with glucose, as indicated by the similar values found for the a nity of the transporter to labelled xylose (K m = 51.9 þ 9.4 mm) and the inhibition by xylose of labelled glucose uptake (K i = 54.0 þ 4.0 mm). These high-capacity transport systems enable C. intermedia to metabolise xylose with the same rate as glucose. In xylose medium, C. intermedia (W max = 0.49 h 31 )compares well to either P. stipitis (W max = 0.44 h 31 [34]) orc. shehatae (W max = 0.38 h 31 [26]). In the low-dilution-rate sugar-limited chemostat cultures glucose was more e ciently utilised by C. intermedia PYCC 4715 than xylose, as evidenced by the lower residual sugar concentrations. This might be the result of the lower K m of the high-a nity glucose transport system and the higher biomass yield (0.51 g g 31 against 0.45 g g 31 in xylose medium), which is possibly due to the excretion of low amounts of xylitol when xylose is used as the carbon source (not shown). However, these values are somewhat questionable because this strain has a tendency to form pseudohyphae and adhere to the fermenter walls. Our results indicate that C. intermedia PYCC 4715 is perhaps the most e cient xylose-utilising yeast under aerobic conditions so far studied, and its xylose metabolism pathway deserves further attention. The rst enzyme of the xylose metabolism pathway (XR) from a di erent C. intermedia strain has been recently characterised [35,36]. It has been found to express two di erent XRs, one (ALR1) being speci c for NADPH whereas the other (ALR2) accepts both NADPH and NADH as cofactors. No biochemical data on the lower xylose metabolism pathway are yet available. As shown, the high-a nity proton symport activity displays an approximately 7-fold increase in cells cultivated under either xylose or glucose limitation. This might be on account of a much higher transporter concentration in the plasma membrane, which would facilitate the isolation of the respective gene(s) and the subsequent molecular studies. 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